IFN-?– and IL-10–expressing virus epitope-specific Foxp3(+) T reg cells in the central nervous system during encephalomyelitis

Zhao, Jingxian; Zhao, Jincun; Fett, Craig; Trandem, Kathryn; Fleming, Erica; Perlman, Stanley

Detection of M133-specific T reg cells in naive T cell pool. M133specific T reg cells in the naive T cell pool were identified using a previously described method (Moon et al., 2009) with modifications. In brief, single cell suspensions were prepared from spleens and cervical, axillary, brachial inguinal, and mesenteric LNs of uninfected 10-wk Foxp3 gfp and OT-II mice. CD4 T cells were isolated by negative selection using a CD4 T cell isolation kit II. CD4 T cells were then stained with anti-CD16/32 mAb (2.4G2) and PEconjugated I-A b /M133 tetramers as described in the previous section (except tetramer was used at 4 µg/ml to reduce background). After washing, cells were exposed to anti-PE magnetic microbeads and separated on an MS column (Miltenyi Biotec). Cells were then stained with anti-CD3-PerCP, anti-CD4-Alexa Fluor 700, and non-T cell lineage (dump) markers (eFluor 450-conjugated anti-CD11b, anti-CD11c, anti-F4/80, anti-B220, and anti-CD49b). Cells were then stained with Hoechst 33258 to gate out dead cells, and analyzed with a LSRII cytometer (BD).